Solution to the 50-year-old Okazaki-fragment problem

Introduction

• DNA has an antiparallel double-stranded structure.
• DNA polymerases work in a 5' to 3' direction, leading to replication of strands in opposite directions.
• Leading strand: synthesized continuously in the direction of replication fork.
• Lagging strand: synthesized discontinuously in fragments (Okazaki fragments).

Historical Background

• Reiji and Tsuneko Okazaki proposed in 1968 that the lagging strand is replicated in small fragments.
• These fragments, named Okazaki fragments, are matured into a continuous strand.

Semidiscontinuous Replication Model

• Textbook model: continuous leading strand and discontinuous lagging strand.
• Original experimental results indicated all nascent DNA fragments are small, suggesting possible discontinuous synthesis of the leading strand.

Recent Findings by Cronan et al.

• Leading strand is actually synthesized continuously, but fragmented by ribonucleotide excision repair (RER).
• RER removes ribonucleotides mistakenly inserted by DNA polymerases.

Experimental Techniques

• Pulse-labeling techniques were used to detect early lagging-strand pieces.
• Okazaki's original experiments showed labeled DNA fragments were small (1 to 2 kb), contradicting the idea of a continuous leading strand.

Improvements and Discoveries

• Okazaki team's advancements:
  • Demonstrated small fragments as intermediates in DNA synthesis.
  • RNase H2 involved in the excision of ribonucleotides.

Ribonucleotide Excision Repair (RER)

• Essential in removing ribonucleotides from DNA.
• Misincorporation due to high intracellular rNTP levels.
• RNase H2 initiates RER by cutting at 5' ribonucleotide positions.

Impact of Okazaki's Work

• Introduced concepts of RNA priming and Okazaki fragment maturation.
• Reaffirmed the semidiscontinuous replication model.

Cronan et al.'s Contributions

• Used advanced techniques (formamide-urea-sucrose gradients) to show leading strand fragmentation is due to RER.
• Demonstrated that the leading strand is synthesized mostly continuously, yet exhibits discontinuities.
• Leading strand fragmentation relates to various DNA repair pathways and replication fork restart mechanisms.

Continuing Questions

• How continuous is the leading strand synthesis?
• Further investigation needed into replication fork stalling and restart mechanisms.
• Possible involvement of other DNA repair pathways.

Acknowledgments

• Research supported by NIH Grant R35-GM118129.

References

• Extensive references to studies on DNA replication, Okazaki fragments, and DNA repair mechanisms are provided for further reading.